Confirmation of natural hybrids between Gentiana straminea and G. siphonantha (Gentianaceae) based on molecular evidence

A few individuals with intermediate morpho-logy always appeared in the sympatric distributions of Gentiana straminea and G. siphonantha. These intermedi-ate individuals were hypothesized to be the hybrids of two species after a careful evaluation of their morphological characteristics. To test this hypothesis, sequence compar-ison of the internal transcribed spacer (ITS) regions of the nuclear ribosomal and trnS (GCU)-trnG (UCC) inter-genic spacer region of the chloroplast DNA from Gentiana straminea, G. siphonantha and the putative hybrids was performed. The results suggest that most intermediate individuals were the natural hybrids between G. straminea and G. siphonantha. In addition, we exam-ined the sequence variation among the individuals of both parent species and analyzed the possibility leading to the incongruent identification in some individuals based on morphologic and molecular evidences, respectively. The intraspecific diversification of DNA fragments within both parent species and their high variability in hybrid swarms probably resulted from chloroplast genome recombination and incomplete lineage sorting during the early stages of speciation origin of the parent species.     

ITS polymorphism within a single strain ofSclerotium rolfsii

Two morphologically distinct strains, 63–76 and 63H1, were isolated from a protoplast and a hyphal tip of the parentalSclerotium rolfsii strain S-63, respectively. Strains 63–76 and 63H1 showed reduced mycelial growth and lacked clamp connections on hyphae. The two strains also differed from each other and from their parent in RAPD patterns generated by several primers, suggesting that 63–76 and 63H1 were homokaryons isolated from the hetereokaryon S-63. Whereas the parent S-63 belonged to ITS-RFLP group 1, RFLP patterns of internal transcribed spacer (ITS) regions of rDNA of 63–76 and 63H1 were similar to those of ITS-RFLP groups 5 and 3, respectively. The sequence similarity of ITS regions were more than 99% between 63–76 and group 5 strains, 100% between 63H1 and the group 3 strain, and 96.3% between 63–76 and 63H1. Direct sequencing failed in the parental strain S-63. S-63 was considered to contain ITS types of groups 5 and 3.     

大白口蘑分离菌株的DNA鉴定

以松口蘑Tricholoma matsutake子实体为外类群,对大白口蘑T. giganteum 野生子实体及其组织分离菌丝进行ITS序列测序,通过DNAStar软件进行比较分析。结果表明大白口蘑ITS序列长度为589bp,松口蘑ITS序列长度为601bp,ITS1和ITS2呈现不同程度的种间多态性;ITS序列测定证实了大白口蘑野生子实体及其组织分离菌丝的同质性,并且ITS区序列在大白口蘑种内不同菌株间的变异程度很小,表明使用通用引物ITS4和ITS5,通过PCR扩增测序即可用于大白口蘑的种质鉴定。     

大白口蘑分离菌株的DNA鉴定

以松口蘑Tricholoma matsutake子实体为外类群,对大白口蘑T.giganteum野生子实体及其组织分离菌丝进行ITS序列测序,通过DNAStar软件进行比较分析。结果表明大白口蘑ITS序列长度为589bp,松口蘑ITS序列长度为601bp,ITS1和ITS2呈现不同程度的种间多态性;ITS序列测定证实了大白口蘑野生子实体及其组织分离菌丝的同质性,并且ITS区序列在大白口蘑种内不同菌株间的变异程度很小,表明使用通用引物ITS4和ITS5,通过PCR扩增测序即可用于大白口蘑的种质鉴定。     

Phylogeny of the GenusTrichodermaBased on Sequence Analysis of the Internal Transcribed Spacer Region 1 of the rDNA Cluster

Sequences of the internal transcribed spacer region 1 (ITS1) of the ribosomal DNA were used to determine the phylogenetic relationships of species ofTrichodermasect.Pachybasium.To this end, 85 strains—including all the availableex-type strains—were analyzed. Parsimony analysis demonstrated that the section is nonmonophyletic, distributing the 85 strains among three main groups that were supported by bootstrap values. Group A comprises two clades (A1 and A2), with A1 includingT. polysporum, T. piluliferum,andT. minutisporum,while A2 includedT. hamatum, T. pubescens,andT. strigosumin addition to species previously included in sect.Trichoderma(i.e.,T. viride, T. atroviride,andT. koningii). Theex-type strain ofT. fasciculatumformed a separate branch basal to clade A. Clade B contained the sect.PachybasiummembersT. harzianum, T. fertile, T. croceum, T. longipile, T. strictipile, T. tomentosum, T. oblongisporum, T. flavofuscum, T. spirale,and the anamorphs ofHypocrea semiorbisandH. cf. gelatinosa.Sequence differences among clades A1, A2, and B were in the same order of magnitude as between each of them andT. longibrachiatum,which was used as an outgroup in these analyses. Sequence differences within clades A1, A2, and B were considerably smaller: in some cases (i.e.,T. virensandT. flavofuscum; T. strictipileandH. cf. gelatinosa), the ITS1-sequences were identical, suggesting conspecifity. In other cases (e.g.,T. crassumandT. longipile; T. harzianum, T. inhamatum, T. croceum, T. fertile,andH. semiorbis; T. hamatumandT. pubescens;andT. viride, T. atroviride,andT. koningii) differences were in the range of 1–3 nt only, suggesting a very close phylogenetic relationship. The sequence of a previously described aggressive mushroom competitor group ofT. harzianumstrains (Th2) was strikingly different from that of theex-type strain ofT. harzianumand closely related species and is likely to be a separate species.     

A phylogeny of Packera (Senecioneae; asteraceae) based on internal transcribed spacer region sequence data and a broad sampling of outgroups

A phylogeny of Packera is presented based on sequence data from the internal transcribed spacer region of nuclear ribosomal DNA of 26 species (28 populations) of Packera and 23 outgroup taxa, including representatives of all three subtribes of the Senecioneae. The results support a Mexican origin for Packera, with its closest relatives found among Old World taxa in the subtribe Senecioninae, such as Senecio jacobaea and Pericallis. Packera species from the west coast of the United States, previously included in the section Bolanderi of Greenman, are part of a basal assemblage including species of Greenman's Mexican section Sanguisorboidei. The rest of Packera separates into two sister groups, one containing species from the Arizona-New Mexico region and the other containing more geographically diverse taxa. Among the outgroups, New World Senecio species are monophyletic and two Tussilaginoid assemblages are strongly supported; the Tephroseroid group (Tephroseris and Sinosenecio) plus Petasites combine with the Luina complex to form a clade of north temperate taxa, and the four Mexican genera (Psacalium, Robinsonecio, Barkleyanthus, and Pittocaulon) form a monophyletic group.     

蓼属头状蓼组rDNA-ITS的序列扩增及分析

以贵州境内蓼属头状蓼组6种(含1变种)植物为材料,对其rDNA的内转录间隔区(ITS)序列进行PCR扩增,得到6种植物的ITS序列,分别为:赤胫散2个居群(Polygonum runcinatum var.sinense,GenBank登录号FJ606887、FJ648802),平卧蓼(P.strindbergii,GenBank登录号FJ648803 ),尼泊尔蓼(P.nepalense,GenBank登录号FJ648804),羽叶蓼(P.runcinatum,GenBank登录号FJ648805),火炭母(P.chinense,GenBank登录号FJ648806)和头花蓼(P.capitatum,GenBank登录号FJ648807).其中赤胫散与平卧蓼的ITS序列为首次报道.序列分析结果表明,蓼属头状蓼组6种植物ITS序列总长度为661～666 bp,ITS1区序列长度为243～246 bp,5.8 S rDNA区序列长度165 bp,ITS2区序列长度253～258 bp,6种植物的差异主要集中在ITS1和ITS2区.聚类分析显示,6种头状蓼组植物具有共同起源,结果支持赤胫散从羽叶蓼变种上升为独立物种.     

Molecular phylogeny of ectomycorrhizal <Emphasis Type="Italic">Pisolithus</Emphasis> fungi associated with pine,dipterocarp, and eucalyptus trees in Thailand

The phylogenetic relationships among 135 Pisolithus basidiomes and two isolates collected from three pine forests, a pine-dipterocarp forest, two dipterocarp forests, and 29 eucalyptus plantations in Thailand were investigated. Internal transcribed spacer (ITS) polymorphism analyses, including terminal RFLP, divided them into 26 groups. The ITS in a representative basidiome of each group was sequenced, and a phylogenetic analysis was performed. The dendrogram suggested that at least three Pisolithus species are present in Thailand. Pisolithus basidiomes collected in the pine forests and under some Shorea roxburghii trees in a pine-dipterocarp forest corresponded to species 5 as previously described by Martin et al. in 2002. Those collected under S. roxburghii and Dipterocarp alatus trees in the dipterocarp forests did not match any previously reported species. Basidiomes collected from the eucalyptus plantations were all identified as Pisolithus albus.     

基于ITS序列探讨杜鹃属的亚属和组间系统关系

首次报道了 15种杜鹃属 (Rhododendron)植物、1种杜香属 (Ledum)植物和Cassiopefastigiata的内转录间隔区(ITS) (包括 5 .8S)序列。加上从GenBank下载的 13种杜鹃属植物和Bajiariaracemosa的ITS序列 ,以C .fastigiata和B .racemosa为外类群 ,用最大简约法对杜鹃属的亚属和组间的系统关系进行了分析。结果表明 :1)杜鹃属是一个单系类群 ,叶状苞亚属为杜鹃属的基部类群 ;2 )杜香属确应归并到杜鹃属中 ,且与有鳞杜鹃亚属有较近的亲缘关系 ;3)有鳞杜鹃亚属和杜香构成一个单系分支 ,该分支是其余无鳞杜鹃花的姐妹群 ;4 )由无鳞杜鹃花组成的一个分支的内部支持率较低 ,其中常绿杜鹃亚属和映山红亚属均为内部支持率很高的单系类群 ,而羊踯躅亚属和马银花亚属均为多系类群 ;5 )在马银花亚属中 ,长蕊杜鹃组和马银花组均分别得到强烈支持 ,马银花组与异蕊杜鹃亚属可能构成姐妹群关系 ,异蕊杜鹃亚属和马银花组组成的一个分支可能与映山红亚属构成姐妹群关系。     

基于ITS序列探讨杜鹃属的亚属和组间系统关系

首次报道了15种杜鹃属(Rhododendron)植物、1种杜香属(Ledum)植物和Cassiope fastigiata的内转录间隔区(ITS) (包括5.8S)序列.加上从GenBank下载的13种杜鹃属植物和Bajiaria racemosa的ITS序列,以C. fastigiata和B. racemosa为外类群,用最大简约法对杜鹃属的亚属和组间的系统关系进行了分析.结果表明: 1)杜鹃属是一个单系类群,叶状苞亚属为杜鹃属的基部类群; 2)杜香属确应归并到杜鹃属中,且与有鳞杜鹃亚属有较近的亲缘关系; 3)有鳞杜鹃亚属和杜香构成一个单系分支,该分支是其余无鳞杜鹃花的姐妹群; 4)由无鳞杜鹃花组成的一个分支的内部支持率较低,其中常绿杜鹃亚属和映山红亚属均为内部支持率很高的单系类群,而羊踯躅亚属和马银花亚属均为多系类群; 5)在马银花亚属中,长蕊杜鹃组和马银花组均分别得到强烈支持,马银花组与异蕊杜鹃亚属可能构成姐妹群关系,异蕊杜鹃亚属和马银花组组成的一个分支可能与映山红亚属构成姐妹群关系.     

Sensitive and rapid detection of Karenia mikimotoi (Dinophyceae) by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) assay was developed to detect the genomic DNA of Karenia mikimotoi using a set of four specific primers based on a ribosomal DNA internal transcribed spacer (ITS). The sensitivity of this LAMP assay was 100-fold higher than regular PCR, and its specificity was validated using other algae as a comparison. Two visual detection approaches were feasible to interpret the positive or negative results. This technology may have the potential to aid in forecasting red-tides on the scene because of its high sensitivity, specificity and rapid detection.     

Taxonomic position of a Chinese <Emphasis Type=Italic>Pleurotus</Emphasis> “Bai-Ling-Gu”: it belongs to <Emphasis Type=Italic>Pleurotus eryngii</Emphasis> (DC.: Fr.) Quél. and evolved independently in China

The Chinese Bai-Ling-Gu is a mushroom named Pleurotus eryngii var. tuoliensis C.J. Mou. This species has been identified as P. nebrodensis or P. eryngii var. nebrodensis. We examined its taxonomic position by analysis of mating, cultivation, and rDNA sequences, and concluded as follows. (1) Bai-Ling-Gu mated with P. eryngii var. eryngii, and the F₁ and F₂ formed fruit bodies. (2) Bai-Ling-Gu mated with P. eryngii var. ferulae, and the F₁ formed fruit bodies. (3) In the di-mon mating test, P. eryngii var. nebrodensis from Sicily mated with monokaryons of P. eryngii var. eryngii but mated hardly at all with those of Bai-Ling-Gu and P. eryngii var. ferulae. The di-mon mating pattern of Bai-Ling-Gu resembled those of P. eryngii var. ferulae. (4) The partial sequences of rDNA ITS1 and IGS1 from the epitype of P. nebrodensis were identical with those from P. eryngii var. nebrodensis from Sicily but differed from those from Bai-Ling-Gu. (5) The strains of P. eryngii var. eryngii and P. eryngii var. ferulae were in a group, the strains of P. eryngii var. nebrodensis from Sicily were in another group, and the strains of Bai-Ling-Gu were in the other group in both the phylogenetic trees based on the ITS1 and the IGS1 sequences. These results led to the conclusion that Bai-Ling-Gu is a variety of P. eryngii and evolved independently in China. It is satisfactory to identify Bai-Ling-Gu with P. eryngii var. tuoliensis C.J. Mou.     

Genetic relationship of Tricholoma matsutake and T. nauseosum from the Northern Hemisphere based on analyses of ribosomal DNA spacer regions

The genetic relationship among Tricholoma matsutake and T. nauseosum strains collected from various parts of the Northern Hemisphere was investigated using sequence analysis of the rDNA ITS region and PCR-RFLP analysis of the rDNA IGS-1 region. ITS sequence similarity between T. matsutake and T. nauseosum ranged between 98.1% and 100%. The strains of T. matsutake from coniferous forests and those from broad-leaved forests showed more than 99.8% similarity in their ITS sequences. Three distinct RFLP types were detected when IGS-1 regions were digested with Cfr13I. RFLP patterns showed no variability among the strains of T. nauseosum and those of T. matsutake from broad-leaved forests. This pattern corresponded to the dominant RFLP type in the Japanese population of T. matsutake. Thus, strains belonging to this RFLP type are widely distributed throughout East Asia and Europe and associated with many tree species of Pinaceae and Fagaceae. The result suggests that T. matsutake in coniferous and broad-leaved forests and T. nauseosum should be treated as the same species genetically.     

维吾尔药材红豆杉植物基原研究

从分子学角度探明维吾尔药材红豆杉的植物基原,为维吾尔药材标准化与维吾尔药产业化提供科学依据。采用试剂盒法提取红豆杉属植物及药材基因组DNA,PCR扩增ITS2片段,双向测序,应用Mega 6.0软件分析序列,计算种内及种间遗传距离,构建NJ鉴别树。红豆杉属植物ITS2序列长度为229~231bp,GC含量为59.13%~60.26%;西藏红豆杉与红豆杉及南方红豆杉种内种间K2P距离为0,与东北红豆杉、命叶红豆杉及曼地亚红豆杉种间K2P距离分别为0.012、0.013、0.015。NJ树结果表明,西藏红豆杉与红豆杉及南方红豆杉无法区分,与东北红豆杉、命叶红豆杉可区分;曼地亚红豆杉与其母本东北红豆杉聚在一起,无法区分。根据ITS2序列鉴定结果,西藏红豆杉与红豆杉、南方红豆杉可作为药材的同一基原,东北红豆杉可与曼地亚红豆杉作为同一基原,为维吾尔药材的真伪鉴别及质量标准提升提供基原鉴定依据。     

High evolutionary divergence of the 5.8S ribosomal DNA in Mimulus glaucescens (Scrophulariaceae)

Ribosomal DNA sequences for the ITS 1, 5.8S, ITS 2 and adjoining regions of the 18S and 25S were obtained from Mimulus glaucescens (Scrophulariaceae) via cloned PCR products. The spacer sequences were completely unrelated to other plant taxa, although spacer lengths were approximately the same. Interestingly, the Mimulus 5.8S sequence was much more divergent than other higher-plant rDNA sequences. Consideration of the secondary structure of the 5.8S rRNA shows that most of the changes in Mimulus are compensatory and preserve the basic secondary structure of the mature RNA molecule.     

洪湖沉积物中真菌群落结构的垂直分布

湖泊沉积物中真菌的类群及其作用所知甚少。以长江中上游代表性湖泊洪湖为研究对象,从湖心截取了一个72cm长的沉积柱,通过rDNA内转录间隔区（ITS）的变性梯度凝胶电泳（DGGE）,对洪湖沉积物中真菌群落结构的垂直变化进行了分析。结果显示,DGGE条带以14－18cm层次为过渡,在2－14cm和14－72cm之间存在一定差异。Shannon-Weaver index（H′）在上下层之间有波动。聚类分析上层2－18cm和中下层18－72cm分别以不同的小类聚在一起。表明14－18cm以上和以下的类群存在差异,